In subjects affected by arterial hypertension, the left ventricle of the heart is subjected to increased mechanical activity in order to pump blood against increased blood pressure. Under these conditions the heart undergoes a compensatory hypertrophy in which cardiomyocytes increase in size as consequence of increased synthesis and assembly of contractile proteins of actomyosin fibrils.
Although hypertrophy is compensatory and beneficial allowing the generation of more contractile force, under condition of chronic high blood pressure, additional events might occur that either reduce the efficacy of the hypertrophy response or activate additional pathways causing cardiac dilation and progressively leading to heart dysfunction and failure.
The identification of the molecular mechanisms involved in the initial cardiac hypertrophy and in the onset of a subsequent defective cardiomyocytes response is a major challenge of the cardiovascular biology and medicine in these days. In fact, understanding such molecular mechanisms can be of great importance to develop therapeutical strategies aimed to fight congestive heart failure, a pathology that, only in the United States of America, affects more that 400.000 peoples every year.
Considerable efforts have been made in the past decade to identify the molecular mechanisms at the cellular level involved in the hypertrophic response of cardiomyocytes. These studies led to a mechanistic model illustrated in FIG. 10 in which mechanical stretching induced by hemodynamic overload (1) trigger intracellular mechanosensors (2) that activate intracellular signaling pathways (3) leading to hypertrophy by dual modes: by direct activation of muscle specific genes (4a) and by inducing secretion of neurohumoral and autocrine factors (4b) that in turn act on the cardiomyocytes via specific receptors and signaling contributing to the hypertrophic response.
Among the signaling molecules (point 3 of FIG. 10) thought to be involved in the cardiac hypertrophy in response to mechanical overload are: the alfa Gq subunit of the heterotrimeric G protein coupled to the beta adrenergic receptors (Akhter et al. 1998), the phospholipase C beta and protein kinase C, acting downstream of the G proteins (Wakasaki et al. 1997), the Calcineurin/NF-AT3 pathway, the Ras cascade including Raf-1 and ERK1/2 MAP kinases, the stress kinases Jnk and p38, the phosphoinositide 3-kinase, the Jak-STAT pathway (for review see Aoki and Izumo 2001; Ruwhof and van der Laarse 2000; Hunter and Chien 1999). These molecules, although very important in inducing the hypertrophic response, are all acting quite downstream along the signaling pathways.
It is thus clear that identification of the mechanosensors themselves (point 2 of FIG. 10) would be of great importance, since interference with such upstream regulatory elements would allow a much more specific control of the hypertrophic response.
The mechanical tension in the muscle is exerted by the contractile proteins of the cytoskeleton, the actomyosin fibrils which are physically linked to the plasma membrane and to the extracellular matrix, via membrane receptors belonging to the integrin family.
In muscles, integrins are preferentially localized in specific sites known as myotendinous junction and costamers. These are specific sites were actomyosin fibrils are connected to the plasma membrane contributing to a correct and stable association of the contractile machinery to the membrane of the muscle cells.
Besides transmitting the contractile force across the plasma membrane, these junctions are also important mechanosensors capable of transmitting signals inside the cell in response to mechanical stretching. Several proteins are in fact localized at these sites at the cytoplasmic face of the plasma membrane and interacting with integrins. These proteins include paxillin, vinculin, talin, and the tyrosine kinase p125Fak. This molecular machinery is activated by mechanical stretching of the cells (for review see Davis et al. 2001; Carson and Wei 2000) and is the best candidate as the mechanosensing apparatus.
A beta1 integrin isoform (beta1D) that is specifically expressed in striated cardiac and skeletal muscle has been disclosed (Belkin et al 1996). In association with the alpha7 subunit, beta1D forms an heterodimer a7b1D with receptor activity toward merosin (laminin 2) of the extracellular matrix. Functional analysis indicated that beta1D integrin binds both cytoskeletal elements and extracellular matrix ligands with much higher affinity compared to the beta1A isoform present in all non-muscle tissues (Belkin et al 1997) suggesting that beta1D provides a stable actin-laminin interaction across the plasma membrane necessary to support the mechanical tension during muscle contraction.
To further define the molecular basis of these functional properties the inventors searched for proteins capable to bind to the cytoplasmic domain of beta1D. Using the two-hybrid screening the inventors isolated melusin, a novel protein selectively expressed in skeletal muscle and heart (Brancaccio et al. 1999; GenBank AF140690; GenBank AF140691).
Sequence analysis of melusin indicated the presence in the amino terminal half of the protein of a tandem repeated cysteine and histidine rich sequence and of putative binding sites for SH2 and SH3 domains. The C terminal half comprises the binding site for the integrin cytoplasmic domain and is characterized by a stretch of acidic amino acid residues binding to Ca2+ (FIG. 1). Melusin is localized at costamers in correspondence of Z line where also integrins and vinculin are concentrated (Brancaccio et al. 1999).
Melusin, thus likely represents a new intracellular transducer of beta1 integrin function in muscle cells.